Sphingolipid Targeting Agent MIPS247 Attenuates Collagen Synthesis by Cardiac and Kidney Cells Via Stabilisation of PPM1A

Background: Cardiac and renal fibrosis is the key pathological process underpinning heart and kidney failure for which there is currently not effective therapy. The pleiotropic roles of sphingolipids have been extensively studied, revealing a janus-faced role in mediating fibrosis. However, the role of enzymes involved sphingolipids synthesis pathways have not been fully investigated. We have recently developed novel sphingolipid targeting agents MIPS247 that perturbs cellular sphingolipid levels. The aim of this study is to determine whether MIPS247 will attenuate collagen synthesis by cardiac and kidney cells and mechanisms involved. Methods: Neonatal rat cardiac fibroblasts (NCF) were isolated from day 1 to 2 pups using enzymatic digestion. Kidney proximal tubule cells, HK2, were purchased from ATCC. All cells were pre-treated with MIPS247 for 2 hours before stimulation with 100 nM Angiotensin II (AngII) or 10 ng/ml TGF for 48 hours. Collagen synthesis were determined by 3H-proline incorporation. Western Blot analysis was used to determine the mechanisms involved. Results: MIPS247 dose-dependently inhibited AngII- and TGFβ-stimulated NCF and HK2 collagen synthesis. Western Blot analysis demonstrated that MIPS247 exerts its effect via protection of a protein phosphatase 2C family member, PPM1A (a key p-SMAD2/3 phosphatase), degradation and inhibition of NFB pathways in NCF (Figure). Conclusion: This is the first demonstration of a small molecule sphingolipid targeting agents MIPS247 attenuating the fibrosis process in both cardiac fibroblasts and kidney cells stimulated by a range of fibrotic factors. Thus, such sphingolipid targeting agents may represent a novel therapeutic agents for cardiac and renal fibrosis.